Biochemical devices and their methods of manufacture

ABSTRACT

Biochemical devices comprising a sensing surface that is at least partially covered by a nanocrystalline metal oxide semiconductor film which provides a recipient surface for immobilizing biochemical species on. The film has a mesoporous surface that gives up to a 1000 increase in biochemical species adsorption when compared to a flat surface. The biochemical devices comprising these surfaces can be optical and electrochemical biosensors and reactors for synthetic or biodegradation reactions.

This application is a national phase of International Application No.PCT/GB99/00999, filed on Mar. 31, 1999 and published in English.

This invention relates to biochemical devices such as biosensors andtheir methods of manufacture.

A wide range of devices used in chemistry and biology (such as in thebiotechnological field) require the immobilisation of a biochemicalspecies upon a substrate or film, so that the biochemical species can besensed or can react with another substance. Such devices includeelectrochemical, optical and electro-optical bioanalytical devices, andreactors for synthetic or biodegradation reactions. These reactors maybe driven optically and/or electrically.

A range of strategies are currently employed for the immobilisation ofbiochemical species in biochemical devices, the strategy used dependingupon the device and its application. For example, an electrochemicalbiosensor device requires electrical contact between the biochemicalspecies, such as a protein, and a conducting electrode. Proceduresemployed in optimising this contact include aligning the biochemicalspecies on chemically modified electrodes, attachingelectron-transporting groups or modified redox co-factors andimmobilising the biochemical in polymer matrices. With opticalbiosensors, however, optical transparency of the solid substrate is akey issue. In these devices polymeric or silicate glass matrices havebeen employed to encapsulate the biochemical species. These biosensorsare used to detect a wide variety of different things, such as sugars orpH.

In many cases, the immobilisation of the biochemical species is achievedduring the process of matrix formation. Such matrix formation requiresdrying for prolonged periods and/or at elevated temperatures. Suchprocedures tend to cause denaturisation of many biochemical species, inparticular proteins.

WO-A-96/00198 discloses a process for producing ceramic layers includingtitanium dioxide. These ceramic layers can have enzymes immobilised inthem for use in the biochemical field. These layers are produced bymixing a suspension containing TiO₂ and an enzyme and then drying it ina stream of warm air at 80° C. Due to the high temperature used indrying this process is only applicable to thermally stable biochemicalspecies. Many biochemical species are not thermally stable.

In accordance with one aspect of the present invention there is provideda biochemical device comprising a surface for immobilising a biochemicalspecies, wherein said surface is at least partially covered with ananocrystalline metal oxide semiconductor film, said film providing arecipient surface for immobilisation of said biochemical species.

Thus the present invention alleviates the disadvantages of the prior artby the use of nanocrystalline metal oxide semiconductor films. Thesefilms typically comprise nanometer-sized crystalline particles (typicaldiameter 5-50 nm) which are densely packed to form a mesoporousstructure with a surface area up to 1000 times greater than itsgeometrical area.

In other words, in embodiments of the invention a biomolecule can beimmobilised on a preformed, mesoporous film, in contrast to previoustechniques of matrix immobilisation where film formation and biomoleculeimmobilisation are achieved in a single process. This allows theimmobilisation to be conducted under conditions which do not denaturethe protein or other biomolecule, of which the use of lower temperaturesis one important example. This combination of mild immobilisationconditions and the specific properties of the film (high biomoleculeloading, optical transparency, stability, electrical conductivity) aretechnical advantages of this invention.

Furthermore, these nanocrystalline metal oxide semiconductor films havesimple and flexible biochemical attachment chemistries. Attachment mayoccur covalently, by adsorption, by bio-derivitisation of the film or bycombinations thereof.

Nanocrystalline metal oxide semiconductor films combine a high surfacearea and excellent stability with efficient current transport Inaddition to a high surface area these substances have rapid diffusionpaths due to the pores being larger than the biochemical species, andthis in turn allows the rapid mass transport of the analyte into andthrough the film. Furthermore, their high surface area to geometricalarea enables a small device to hold a large quantity of the biochemicalspecies. This enables the size of the device to be decreased resultingin increased mass transport giving faster response times. Additionally asmaller device has the practical advantage of being able to be used inrestricted volumes. Furthermore, the high loading of the biochemicalmolecules makes them less susceptible to loss of activity. No existingmaterials employed for biochemical applications exhibit all of theseproperties.

In a preferred embodiment the nanocrystalline metal oxide semiconductoris titanium dioxide, TiO₂. TiO₂ has a wide band gap and as such isoptically transparent, making it suitable for optical applications aswell as electrical ones.

In further embodiments the nanocrystalline metal oxide semiconductor iszinc oxide, ZnO or zirconium dioxide, ZrO₂.

In one embodiment a biochemical species is immobilised on at least aportion of the film. Preferably, the biochemical species is a protein.The term protein, when used in this application should be taken toinclude enzymes, antibodies or fragments thereof and other polypeptidescapable of binding molecules or catalysing their transformation toanother molecular species.

In a further embodiment attachment of the biochemical species occurs bybio-derivitisation of the film. Bio-derivitisation of the film involvesusing a chemical species —such as a biomolecule—as an intermediary, thebiochemical species molecule becoming immobilised to the film via thechemical species—i.e. the biochemical species is bound to the chemicalspecies which is in turn bound to the film. This has the advantage ofincreasing the number of possible biochemical species that can beimmobilised by the film, and improving the stability of immobilisation.An example of this is the use of the enzyme avidin. Avidin is expectedto bind strongly to TiO₂ due to its positive charge. Any biomoleculewith a biotin group attached (readily added) can bind to avidin.

In a further embodiment the biochemical device is a biosensor. Thenanocrystalline metal oxide semiconductor film providing an idealsurface for inmmobilising a sensing biochemical species for use in sucha device.

Advantageously, the film forms an array on the surface. A convenientlyshaped sensing area can thus be formed. Furthermore, the array allowsfor different sensing biochemical species to be attached to differentportions of the array. Thus a variety of substances can be detected anddepending on the biochemical species used, both electrochemical andoptical signals may be produced. These arrays may be simply andaccurately produced by screen printing or other method compatible withthe properties of the substance being deposited.

In a preferred embodiment a pH sensitive dye is additionally attached toa further portion of the film. Thus changes in the sample pH can bemonitored optically and the results can be used to correct for pHeffects on, for example, an enzyme-based sensing element.

In one embodiment the biosensor is an electrochemical biosensor,comprising an electrical circuit connected to the film, the circuitcomprising a meter for monitoring changes in the current, voltage,conductivity or impedance in the circuit produced by an electrochemicalreaction. The conductive nature of the film makes it especially suitedto such a device.

In a further embodiment the biosensor comprises an optical sensor thatacts to optically detect substances, by monitoring the interaction ofelectromagnetic radiation with the molecules present. The transparentnature of many metal oxide semiconductor films makes them particularlysuited to such an application. In preferred embodiments the immobilisedbiochemical species is a fluorescent labelled or fluorophore labelledbiochemical species and it is the fluorescence thus produced thatprovides an indication of the concentration of the substance underinvestigation. Control electronics form part of the device and are usedto calculate the concentration. Alternatively, the fluorescence mayarise from the binding of a fluorescent molecule to a biochemicalspecies already on the surface. Fluorescence may also be generated bythe formation of a fluorescent product from a non-fluorescent substratethrough an enzymatic reaction.

In one embodiment the device comprises both an electrochemical biosensorand an optical one, such that a plurality of substances may be detectedby the one sensor. The conductive and transparent nature of the filmmakes it particularly suitable for use in such a dual purposeenvironment.

In a further preferred embodiment the sensing biochemical species can beelectrochemically or photochemically switched to a reactive state byoxidation or reduction or through the production of small molecules orions e.g. H⁺. This allows the sensing element in the device to beregenerated by switching the direction of the electric current afteroptical sensing. Furthermore, where there are concerns about thestability of the sensing molecule the current measured during theelectrochemical regeneration step would be an indication of the amountof active material present and so give an opportunity for recalibration.

In a preferred embodiment the biosensor comprises a photoelectricelement or other power generating element such that the biosensor can beused in remote areas, for military applications and for long termsensing, with data being sent by telemetry. Advantageously thephotoelectric element may be a portion of the TiO₂ film, itsphotovoltaic properties acting to produce the necessary power.

In a further embodiment the biochemical device is a reactor forsynthetic, catalytic or biodegradation reactions. The film provides asuitable site for immobilising biochemical species, in particular,enzymes involved in the reaction. In one embodiment the device comprisesan electrical source for electrically driving the reaction, theelectrical conductivity of the film making it particularly suitable forsuch an arrangement In another embodiment the biochemical devicecomprises an optical source, the reaction being driven optically. Thetransparent nature of many nanocrystalline metal oxide semiconductorfilms makes them particularly suited to such an application.

In a preferred embodiment the biochemical reactor comprises aphotoelectric element for producing the reaction driving current.Advantageously the nanocrystalline metal oxide semiconductor film may beTiO₂, and the photoelectric element may be a portion of that TiO₂ film,its photovoltaic properties acting to produce a photoelectric current.

In some embodiments the reaction may be optically driven, thebiochemical device being arranged to receive electromagnetic radiation,possibly by the provision of optically transparent windows in the outercasing of the biochemical device. Alternatively, the biochemical devicemay include a light source.

In accordance with another aspect of the present invention there isprovided a method of manufacturing a biochemical device, comprisingcovering at least a portion of a sensing surface with a film ofnanocrystalline semiconductor, contacting said film with a biochemicalspecies such that said biochemical species is immobilised onto saidfilm. The immobilisation is preferably achieved after fabrication of thesemiconductor film, under conditions which may be selected to minimiseor at least reduce degradation/denaturisation of the biochemicalspecies.

Preferably, the film is applied by screen printing followed by sintetingin air. Nanocrystalline metal oxide semiconductors are particularlysuited to screen printing as they form colloidal suspensions. Screenprinting is a well established and cheap technology providing films fromlow cost precursors. Such means of fabrication also enables robust filmsof the material to be deposited in various patterns.

In some embodiments the biochemical species are caused to contact thepreformed film by immersing said at least partially covered sensingsurface into an aqueous solution of a biochemical species such that saidbiochemical species is immobilised onto said film. This immobilisationmay be achieved without the use of non-physiological temperatures, pHand solvents.

The generic nature of the immobilisation chemistry onto thenanocrystalline material, in particular through adsorption or covalentattachment, means the deposition of the biochemical species canadvantageously be done using a commercially available “gridding robot”.This is an instrument which allows volumes of liquids to be dispensed atspecified x-y co-ordinates. Different liquids (e.g. biomolecules insolution) can be dispensed in an arbitrary pattern. The advantage isthat once the pattern of sensing elements has been laid down by printingthe biomolecules can be patterned on top using the robot. In alternativeembodiments other deposition methods such as ink jet printing may beused.

In preferred embodiments the temperature at which the film is contactedwith the biochemical species is 4° C., in order to optimise stability ofthe biochemical species.

In some embodiments the biochemical species is a protein.

Embodiments of the present invention will now be described, by way ofexample only, and with reference to the accompanying drawings, in which:

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates a biosensor according to an embodiment of the presentinvention;

FIG. 2 illustrates the fluorescent emission spectra of IANBD labelledmaltose binding protein coated TiO₂ films immersed in maltose andsucrose solutions;

FIG. 3 illustrates an electro-optical biosensor;

FIG. 4 illustrates absorption spectra of cytochrome c coated TiO₂ filmsbefore and after application of −0.6V vs Ag/Ag cl (reference electrode):and

FIG. 5 illustrates photochemical reduction of cytochrome-c showingabsorption spectra of cytochrome C/TiO₂ films before (−) and after ( . .. . ) ultraviolet illumination of the film.

FIG. 1 illustrates a fluorescent biosensor for sensing the presence ofmaltose. The biosensor comprises a substrate 10 which is covered by afilm 20 of TiO₂ with a IANBD(4-[N-(2-(iodoacetoxy)ethyl)-N-methylamino]-7-nitobenz-2oxa-1,3diazole)labelled Maltose Binding Protein MBP) immobilised on it, a container forholding the solution 30 under investigation, a light source 40, afluorescence detector 50 and control electronics.

The coated substrate was produced by screen printing a 10μm thicknanocrystalline TiO₂ film onto the substrate using a colloidalsuspension of TiO₂, and then immersing the substrate in an aqueoussolution of a IANBD labelled Maltose Binding Protein (MBP) at 4° C. Thisresults in an approximate monolayer coverage of the film with MBP. Thiscoverage is up to a 1000 fold increase in adsorption relative to a flatsurface due to the mesoporous structure of the film.

The biosensor operates by immersing the MBP covered substrate in thesolution 30 under investigation. Any maltose present in the solutionwill bind to the MBP thereby increasing the fluorescence of the label byup to 200%. The substrate is illuminated by a light source 40 at anappropriate wavelength and the fluorescence is detected by afluorescence detector 50, the detection being aided by the opticaltransparency of the film. Control electronics 60 calculate the amount ofmaltose present in the solution from the fluorescence detected, andoutput the result.

Alternatively, the, generic nature of the immobilisation chemistry ontothe nanocrystalline material, in particular through adsorption orcovalent attachment, means the deposition of the biochemical species canadvantageously be done using a commercially available “gridding robot”.This is an instrument which allows volumes of liquids to be dispensed atspecified x-y co-ordinates, Different liquids (e.g. biomolecules insolution) can be dispensed in an arbitrary pattern. The advantage isthat once the pattern of sensing elements has been laid down by printingthe biomolecules can be patterned on top using the robot. In alternativeembodiments other deposition methods such as ink jet printing may beused.

In other embodiments, a fluorophore-labelled species could be used.

FIG. 2 illustrates the results produced by the device illustrated inFIG. 1, for a solution containing 500 μM maltose and one containingsucrose. As expected the solution containing maltose causes thefluorescence intensity to increase, whereas the control solutioncontaining only sucrose shows no change in fluorescence intensity.

FIG. 3 illustrates an electro-optical biosensor, comprising a substrate10 which is covered by a film 20 of TiO₂ with cytochrome c immobilisedon it. The substrate is connected to a variable voltage supply 70. Anabsorption spectrometer is shown schematically as a light source 45 anda detector 55, but the actual implementation of such a spectrometer toprovide an absorption spectrum through the biosensor is well known inthe art. A detector can be connected in the circuit to monitor chancesin the current and/or voltage in the circuit produced by anelectrochemical reaction taking place.

The absorption spectra illustrated in FIG. 4 are produced by the deviceillustrated in FIG. 3. The two spectra are produced by the cytochrome cbefore and after the application of −0.6V to the back surface of thesubstrate respectively. The cytochrome c protein was immobilised on theTiO₂ coated substrate by immersion of the substrate in an aqueoussolution of cytochrome c at 4° C.

FIG. 4 shows changes in the characteristic reduction of the cytochrome cwith applied voltage and it is therefore clear that there is electricalconnectivity between the external circuit and the adsorbed protein.These results thereby demonstrate the suitability of a substrate coatedwith a TiO₂ film for use in an electrochemical biosensor.

Furthermore, such a biosensor can be electrochemically switched to areactive state by an applied voltage that aids oxidation or reduction.This allows the sensing element in the device to be regenerated byswitching the direction of the electric current after optical sensing.It has further been demonstrated that the immobilised cytochrome c maybe reduced by ultraviolet illumination. Illumation was achieved by 337nm pulses from a nitrogen laser, resulting in band gap excitation of theTiO₂ film. Thus the redox states of immobilised proteins may bemodulated by electromagnetic illumination, resulting from either aseparate photoelectric element or from solar irradiation. Thus thesensing element in the device may be regenerated by either electricalcurrent or electromagnetic irradiation. Such reduction/oxidation of thebiomolecule is also applicable to the function ofelectrocatalytic/photocatalytic biochemical devices for synthetic,bioremediation and other biotechnological applications.

FIG. 5 illustrates the photochemical reduction of Fe(III) Cyt-c toFe(II) Cyt-c driven by 337 nm bandgap excitation of the TiO₂. Thisreduction is attributed to photoinduced electron transfer from theconduction band of the TiO₂ to the immobilised protein. Thisdemonstrates that TiO₂/biomolecule devices arranged forsynthetic/catalytic/biodegradation reactions can be driven optically aswell as electrically. This photochemical reduction (or, in principleoxidation) could also be used to regenerate the sensing state duringbiosensor function.

Table 1, below, gives a summary of some of the proteins that have beensuccessfully immobilised on TiO₂ nanoporous films.

TABLE 1 Protein Source Activity Comments Activity Cytochrome c MammalianElectron transfer Electrochemically coupled ✓ to TiO₂ Maltose bindingBacteria ligand binding Ligand binding detected by ✓ protein(recombinant) fluorescence Cytochrome c Yeast Hydrogen peroxide Nottested peroxidase (recombinant) reduction Haemoglobin Mammalian O₂binding Electrochemically coupled ✓ to TiO₂. Can be used as an NOsensor. Alkaline Bacteria Hydrolysis of Activity measured by ✓phosphatase (recombinant) Phosphate esters fluorescence. Both wild- typeand “his tag” proteins can be immobilised. The latter after Ni²⁺treatment Horseradish Plant Substrate oxidation Activity measure by ✓peroxidase absorbance

The above examples illustrate a key advantage of using a substratecoated with a TiO₂ film, namely that immobilisation of the biochemicalspecies is achieved at 4° C., thereby reducing the risks ofdenaturisation. Another advantage of using a TiO₂ film is that in someembodiments a portion of the film may be used as a photoelectricelement, allowing the device to be used in remote locations without aseparate power source.

In other embodiments of the present invention other nanocrystallinesemiconductor films, such as ZnO or ZrO are used.

In further embodiments the nanocrystalline metal oxide semiconductorfilm is in the form of an array which is screen printed onto thesurface. Different biochemical species may be attached to differentportions of the array and in some embodiments a pH sensitive dye is alsoapplied to the surface.

Other embodiments of the present invention include the use ofnanocrystalline semiconductor metal oxide films in reactors forsynthetic or biodegradation reactions. These reactors can beelectrically or optically driven.

In further embodiments a pH sensitive dye is additionally attached to afurther portion of the film. Thus changes in the sample pH can bemonitored optically and the results can be used to correct for pHeffects on, for example, an enzyme-based sensing element.

It will be apparent, of course, that the present invention has beendescribed above by way of example only and that modifications may bemade within the scope of the appended claims.

1. A biosensor for detecting an analyte of interest, comprising asurface, a preformed nanocrystalline metal oxide semiconductor film atleast partially covering said surface and at least onetemperature-sensitive protein that would be denatured if subjected tonon-physiological temperatures immobilized on at least a portion of saidpreformed film without the use of non-physiological temperatures, suchthat the biosensor will detect the analyte.
 2. A biosensor according toclaim 1, wherein said nanocrystalline metal oxide is titanium dioxide.3. A biosensor according to claim 1, wherein said nanocrystalline metaloxide is zinc oxide.
 4. A biosensor according to claim 1, wherein saidnanocrystalline metal oxide is zirconium dioxide.
 5. A biosensoraccording to claim 1, wherein said film is a bioderivitised film towhich said at least one protein is immobilised.
 6. A biosensor accordingto claim 1, wherein said film forms an array on said surface.
 7. Abiosensor according to claim 6, wherein different proteins are bound todifferent portions of said array.
 8. A biosensor for detecting ananalyte of interest, comprising a surface, a nanocrystalline metal oxidesemiconductor film at least partially covering said surface and at leastone protein immobilized on at least a portion of said film without theuse of non-physiological temperatures, such that the biosensor willdetect the analyte, and further comprising a pH-sensitive dye partiallycovering said surface.
 9. A biosensor according to claim 1, wherein saidbiosensor is an electrochemical biosensor, and further comprising anelectrical circuit electrically connected to said film, said circuitcomprising a detector for monitoring changes in the current or voltagein said circuit produced by an electrochemical reaction.
 10. A biosensoraccording to claim 1, wherein said biosensor is an optical biosensor,and further comprising an optical sensor for monitoring a reaction bysensing the interaction of electromagnetic radiation with the moleculespresent.
 11. An optical biosensor according to claim 10, wherein said atleast one protein is a fluorescent or fluorophore labelled protein, saidfilm is optically transparent, and further comprising a light source andcontrol electronics for calculating concentrations from the output ofsaid optical sensor.
 12. A biosensor according to claim 1, furthercomprising an electrical circuit electrically connected to said film,and an optical sensor.
 13. A biosensor according to claim 12, whereinsaid at least one protein is such as to be electrochemically orphotochemically switched to a sensing state by oxidation or reduction,the results of the sensing reaction being measured optically orelectrically.
 14. A biosensor according to claim 1, wherein saidbiosensor further comprises a power supplying element.
 15. A biosensoraccording to claim 14, wherein said power supplying element comprises aphotoelectric element operable to supply power in response toelectromagnetic radiation.
 16. A biosensor according to claim 15,wherein a portion of said film forms said photoelectric element.
 17. Amethod of manufacturing a biosensor for detecting an analyte ofinterest, comprising the steps of covering at least a portion of asurface with a film of a nanocrystalline metal oxide semiconductor,contacting said preformed film with at least one temperature sensitiveprotein that would be denatured if subjected to non-physiologicaltemperatures, to immobilize said protein on said preformed film withoutthe use of non-physiological temperatures, such that the biosensor willbe operative to detect the analyte.
 18. A method of manufacturing abiosensor according to claim 17, wherein said film is applied to saidsurface by screen printing.
 19. A method of manufacturing a biosensoraccording to claim 17, wherein said film is contacted with a protein byimmersion of said at least partially covered surface in an aqueoussolution of said protein.
 20. A method of manufacturing a biosensoraccording to claim 17, wherein said protein is deposited on said filmusing a gridding robot or other dispensing device such as an ink-jetprinter.
 21. A method of manufacturing a biosensor according to claim17, wherein the temperature at which said film is contacted with saidprotein is substantially 4° C.